Harshe et al. · Nature Communications · 2020

Endogenous antisense RNA curbs CD39 expression in Crohn’s disease

Harshe RP, Xie A, Vuerich M, Frank LA, Gromova B, Zhang H, Robles RJ, Mukherjee S, Csizmadia E, Kokkotou E, Cheifetz AS, Moss AC, Kota SK, Robson SC, Longhi MS

Read the paper The product used

The study

What was asked, and what was found

Harshe et al., Nature Communications, 2020 asked whether CD39, the ectonucleotidase that regulatory T cells and a subset of T helper 17 cells use to hydrolyse extracellular ATP, is held down by an antisense transcript read from its own locus. Mining the human 10q24.1 region, the authors found a long non-coding RNA in antisense orientation to CD39/ENTPD1, ENTPD1-AS1, and confirmed two splice variants by RT-qPCR. The transcript was enriched in regulatory T cells and T helper 17 cells from people with Crohn's disease, while CD39 messenger RNA in the same cells was reduced, and its level tracked Montreal type in regulatory T cells and the Harvey-Bradshaw index in T helper 17 cells.

To test whether the transcript controls CD39, the group silenced it with self-delivering AUMlnc sdASOs, two of them aimed at separate splice variants, added to the medium at 10-20 μM for 72 hours with no transfection reagent. In Jurkat cells the antisense RNA fell across the 72 hour window and CD39 messenger RNA rose. In regulatory T cells and T helper 17 cells from both healthy donors and patients the same exposure lowered the antisense RNA and raised CD39 messenger RNA, the frequency of CD39 positive cells and CD39 mean fluorescence intensity, and patient regulatory T cells suppressed CD4+CD25- proliferation better afterwards. T helper 1 and T helper 2 cells, which carry little of the transcript, did not respond, and FOXP3 was unchanged.

The study then moved in vivo. NOD/scid/gamma mice reconstituted with human CD4 T cells carrying the transcript were given a single enema of trinitrobenzene sulfonic acid and, at the same time, a single intraperitoneal dose of 5.4 mg/kg, then assessed 72 hours later. Treated animals had a lower disease activity index (P = 0.035), greater colon length (P = 0.04) and a lower histology score (P = 0.002) than vehicle, with more CD4 positive CD39 positive cells in blood (P = 0.048) and fewer interleukin 17 producing CD4 cells (P = 0.049). RNA pulldown with mass spectrometry named nucleolin and heterogeneous nuclear ribonucleoprotein A1 as the most abundant proteins bound by the transcript, and silencing either raised CD39 in patient cells.

Key findings

An antisense long non-coding RNA read from the 3 prime end of the human CD39 locus holds CD39 down, and it is raised in regulatory T cells and T helper 17 cells from people with Crohn's disease while CD39 messenger RNA in the same cells is lowered.(Results, High CD39-AS levels in Crohn’s derived Treg and Th17 cells)
Silencing that transcript with AUMlnc sdASOs, added to the medium with no transfection reagent, lowered the antisense RNA across 72 hours and raised CD39 messenger RNA in Jurkat cells.(Results, Endogenous CD39-AS RNA regulates the expression of CD39)
The same treatment raised CD39 messenger RNA, the frequency of CD39 positive cells and CD39 mean fluorescence intensity in regulatory T cells and T helper 17 cells taken from patients.(Results, Endogenous CD39-AS RNA regulates the expression of CD39)
Regulatory T cells from patients suppressed better after the antisense transcript was silenced.(Results, Endogenous CD39-AS RNA regulates the expression of CD39)
In humanized NOD/scid/gamma mice given trinitrobenzene sulfonic acid, a single intraperitoneal dose lowered the disease activity index and the histology score and left the colon longer than vehicle.(Results, Blockade of CD39-AS RNA ameliorates experimental colitis)
The antisense transcript sits mainly in the nucleus and binds nucleolin and heterogeneous nuclear ribonucleoprotein A1, and silencing either protein raises CD39.(Results, CD39-AS interacts with NCL and HNRNPA1)

Product: AUMlnc sdASO
Targets: ENTPD1-AS1 · human · long non-coding RNAENTPD1-AS1 · human · long non-coding RNA, splice variants v1 and v2ENTPD1 · human · mRNA, readout only
Models: Jurkat human T cell line · cell linehuman regulatory T cells and T helper 17 cells polarised from peripheral blood and lamina propria CD4 cells of Crohn's disease patients and healthy donors · primary cellsNOD/scid/gamma mice reconstituted with human CD4 T cells, trinitrobenzene sulfonic acid colitis, single intraperitoneal injection · in vivo
Delivery: added to the culture medium at 10-20 μM for 72 h with no transfection reagent; in vivo a single intraperitoneal injection at 5.4 mg/kg
Readouts: RT-qPCR, flow cytometry, BaseScope and RNAScope in situ hybridization, 3H-thymidine suppression assay, RNA pulldown with mass spectrometry, subcellular fractionation, disease activity index, colon length, histology score
Measured: in vitro concentration and exposure: 10-20 μM, 72 h (Jurkat, regulatory T and T helper 17 cells)in vivo dose: 5.4 mg/kg (single intraperitoneal injection at the time of trinitrobenzene sulfonic acid administration)concentration used to pre-treat regulatory T cells before transfer: 10 μM (sorted CD4+CD25highCD127low regulatory T cells, before intraperitoneal injection)fall in antisense RNA and rise in CD39 mRNA in Jurkat cells: a: **P = 0.002, ##P = 0.001; b: *P = 0.046 (n = 4 replicates, oligonucleotides against the two variants pooled)disease activity index in treated against vehicle mice: *P = 0.035 (vehicle n = 5, treated n = 6, at harvest 72 h after trinitrobenzene sulfonic acid)colon length in treated against vehicle mice: *P = 0.04 (vehicle n = 5, treated n = 6)colon histology score in treated against vehicle mice: **P = 0.002 (haematoxylin and eosin stained colon sections at harvest)frequency of CD4 positive CD39 positive cells and of interleukin 17 producing CD4 cells in peripheral blood: *P = 0.048 for CD4+CD39+, *P = 0.049 for CD4+IL-17+ (vehicle n = 5, treated n = 6, at harvest)
Figures: Fig. 1 The antisense transcript sits at the 3 prime end of the human CD39 locus, and cell lines with high antisense RNA carry low CD39 messenger RNA.Fig. 2 The antisense transcript is raised, and CD39 messenger RNA lowered, in regulatory T cells and T helper 17 cells from Crohn's disease patients.Fig. 3 Silencing the antisense transcript raises CD39 messenger RNA and CD39 mean fluorescence intensity and improves regulatory T cell suppression.Fig. 4 A single intraperitoneal dose in humanized NOD/scid/gamma mice lowers disease activity index and histology score and preserves colon length.Fig. 5 The antisense transcript is nuclear and binds nucleolin and heterogeneous nuclear ribonucleoprotein A1.Fig. 6 Silencing nucleolin, alone or with heterogeneous nuclear ribonucleoprotein A1, raises CD39 in patient regulatory T cells and T helper 17 cells.
Citation: Harshe et al. Endogenous antisense RNA curbs CD39 expression in Crohn’s disease. Nature Communications 2020;11:5894. doi:10.1038/s41467-020-19692-y

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